PROJECT SUMMARY Arsenic has been identified as a prominent causal agent in skin, lung, bladder, and liver cancers. Arsenic contamination impacts hundreds of millions of people in the world. The carcinogenicity of arsenic coupled with an alarmingly large number of people exposed creates an urgency for studying and understanding its carcinogenic mechanisms so effective therapeutic intervention can be initiated. Unlike most other genes, canonical histone messenger RNAs (mRNAs) such as histone H3.1 mRNA do not end with a poly(A) tail, instead they have a stem-loop structure at their 3? end. Stem-loop binding protein (SLBP) attaches to the stem- loop RNA structure that is required for 3?-processing of the canonical histone mRNA. Previously we found that arsenic exposure downregulates SLBP levels, allowing the canonical histone H3.1 mRNA to acquire a poly(A) tail. Polyadenylation of H3.1 mRNA appeared to be carcinogenic, since it induced transcriptional deregulation, cell cycle arrest, and genomic instability, and facilitated anchorage-independent cell growth and tumor formation in nude mice. These effects were likely resulting from disruption of variant histone H3.3 assembly, because genome-wide histone mapping showed that polyadenylation of H3.1 mRNA compromised the H3.3 assembly at the sites critical for transcription, cell identity, and heterochromatin spreading. H3.3 plays important roles in transcription, efficient DNA damage repair, proper segregation of chromosomes, and development. The knockdown of H3.3, which mimics disruption of H3.3 assembly, induced cell transformation. Furthermore, H3.3 mutants have been linked to various type of cancers, underscoring importance of H3.3 in carcinogenesis. Based on these observations, we hypothesize that disruption of H3.3 assembly resulting from polyadenylation of canonical histone H3.1 mRNA is a significant contributor to arsenic-induced carcinogenesis. To test this hypothesis, we will determine the mechanisms by which polyadenylated canonical histone H3.1 mRNA disrupts assembly of the variant H3.3 in Aim 1, determine whether defective H3.3 assembly is responsible for arsenic-induced aberrant transcription, cell cycle arrest as well as genomic instability in Aim 2, and determine the role for disruption of H3.3 assembly in arsenic-induced cell transformation and explore whether arsenic exposure disrupts H3.3 assembly in vivo in Aim 3. The significance and innovation of these studies lie in the potential to reveal disruption of H3.3 assembly as a novel mechanism of arsenic-induced carcinogenesis.